Characterization and functional expression of the cDNA encoding human brain quinolinate phosphoribosyltransferase.
Mammalian quinolinate phosphoribosyltransferase (QPRTase) (EC 2.4.2.19) is a key enzyme in catabolism of quinolinate, an intermediate in the tryptophan-nicotinamide adenine dinucleotide (NAD) pathway. Quinolinate acts as a most potent endogenous exitotoxin to neurons. Elevation of quinolinate levels in the brain has been linked to the pathogenesis of neurodegenerative disorders. As the first step to elucidate molecular basis underlying the quinolinate metabolism, the cDNA encoding human brain QPRTase was cloned and characterized. Utilizing partial amino acid sequences obtained from highly purified porcine kidney QPRTase, a human isolog was obtained from a human brain cDNA library. The cDNA encodes a open reading frame of 297 amino acids, and shares 30 to 40% identity with those of bacterial QPRTases. To confirm that the cDNA clone encodes human QPRTase, its functional expression was studied in a bacterial host. Introduction of the human cDNA into a QPRTase defective (nadC) E. coli strain brought about an abrupt increase in QPRTase activity and allowed the cells to grow in the absence of nicotinic acid. It is concluded that the cloned cDNA encodes human QPRTase which is functional beyond the phylogenic boundary.
Fukuoka
S I
SI
Research Institute for Food Science, Kyoto University Uji, Japan. fukuoka@food2.food.kyoto-u.ac.jp
Nyaruhucha
C M
CM
Shibata
K
K
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D005816:Genetic Complementation Test; D006801:Humans; D007124:Immunoenzyme Techniques; D008969:Molecular Sequence Data; D010430:Pentosyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)
D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D010430:Pentosyltransferases; D000098553:Nicotinate-Nucleotide Diphosphorylase (Carboxylating)